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(n) EP 1 262 764 A1

PATENT APPLICATION

(51) IntClJ: G01N 21/77, G01N 33/50,
G01N 21/64, B01L3/00

Europaisches Patentamt
European Patent Office
Office europeen des brevets

(12) EUROPEAN

(43) Date of publication:

04.12.2002 Bulletin 2002/49

(21) Application number: 01401376.7

(22) Date of filing: 25.05.2001

(84) Designated Contracting States:

AT BE CH CY DE DK ES Fl FR GB GR IE IT LI LU
MC NL PTSETR
Designated Extension States:
AL LT LV MK RO SI

(71) Applicant: CORNING INCORPORATED
Corning, N.Y. 14831 (US)

(72) Inventors:

• Lemee, Valerie J. C.

SP FR 02-12, Corning, NY 14831 (US)

(54)

(57) A system, method and device for the detection
of reactions between biomolecules or cells and a sec-
ond compound are disclosed. The invention utilizes a
fluorescent material having a fluorescence that changes
with temperature. The fluorescent material is associated

• Pecheul,Marylene D. M.

SP FR 02-12, Corning, NY 14831 (US)

• Marque, Pascal

SP FR 02-12, Corning, NY 14831 (US)

• Root, David M.

SP FR 02-12, Corning, NY 14831 (US)

(74) Representative: Marchant, James Ian et al
Elkington and Fife,
Prospect House,
8 Pembroke Road
Sevenoaks, Kent TN13 1XR (GB)

with a substrate, for example, a microarray chip or mi-
croplate, preferably suitable for use in high-throughput
screening of biomolecules or cells. Substrates contain-
ing the fluorescent material also can be used to com-
pensate for temperature variations in refractive index in
optical sensors.

Method and device for the detection of reactions and metabolic changes with
temperature-sensitive fluorescent material

Printed by Jouve. 75001 PARIS (FR)

EP 1 262 764 A1

Description

FIELD OF THE INVENTION

5 [0001] This invention relates to detection of chemical reactions and metabolic changes in biological materials using
fluorescent materials. More particularly, the present invention relates to systems, devices and methods of detecting
reactions involving chemicals, biomolecules and other compounds and metabolic changes in cells, the systems, meth-
ods and devices utilizing a fluorescent material, the fluorescence of which depends on temperature.

10 BACKGROUND OF THE INVENTION

[0002] The drug discovery process is a multiple step process involving identification of disease targets, assay de-
velopment and validation, high throughput primary screening of compound libraries, hit validation in secondary screens,
lead optimization, Absorption Distribution Metabolism and Excretion (ADME) and toxicity in pre-clinical trials. This long

15 process eventually leads to a drug candidate that enters clinical trial phases. The assay development and validation
phase is used to optimize the labeling system and detection method to be used for a robust, low background and low
variability screen. Standard labels are either radioactive elements or fluorescent/luminescent or absorbing compounds.
High throughput screening of compound libraries requires automated parallel handling and processing of labeled bio-
logical reagents and compound mixtures. Standard screens are performed using microarray chips, microfluidic chips,

20 and microtiter plates (hereinafter "microplates") with 96, 384 or 1536 wells compatible with fluid handling equipment
and detection instruments. Recent developments of Charge Coupled Device (CCD) based detection instruments and
compound arraying techniques allow for screening compounds in formats having higher densities than standard mi-
cropiates.

[0003] Cell-based assays are often used in the drug discovery process. They are particularly useful when the drug
25 target is a transmembrane receptor or an ion-channel. Scintillation proximity assays, as well as fluorescence assays
have been designed to monitor the physiological state of the cells, for example, by monitoring the level of second
messenger concentrations (e.g., cAMP, Na + , DAG, etc.). In some cases, a cell lysis step is required for the measure-
ment. In other cases, the cells are transfected with a fluorescent protein, the fluorescent properties of which depend
on the concentration of a second messenger. High content screens also exist in which labeled molecules are used to
30 visualize receptor endocytosis, recycling and intracellular trafficking of messenger biomolecules, providing additional
information on the physiological effects cause by receptor/ligand binding.

[0004] It is advantageous to monitor the cell physiological state without using time consuming and labor intensive
labeling steps such as genetic engineering or even perfusion. An effective way of performing such assays is by meas-
uring acidification rates of the medium in which cells are suspended (J.C. Owicki, J. Wallace Parce, 'Biosensors Based

35 on the Energy Metabolism of Living Cells: The Physical Chemistry and Cell Biology of Extracellular Acidification, Bio-
sensors & Bioelectronics, 1 992, 7, 255-272). Another way of performing such assays involves using a microphysiometer
that uses highly sensitive pH sensors, which is available from Molecular Devices Corp., Sunnyvale, CA. Still another
way of monitoring the physiological state of cultured cells is by measuring the heat flow using a calorimetric technique.
However, none of these label-free technologies is compatible with the requirement of high throughput for screening

40 thousands of compounds from large chemical libraries.

[0005] Imaging infrared thermography is another technology that is used to monitor physiological and molecular
events that elicit a thermogenic response in animals, plants, tissues, cells and cell-free systems. This method can be
used for screening drug candidates. Whereas this method can provide throughput, the detection principle is difficult to
master because it cannot easily produce absolute temperature measurements. Moreover, besides the performance of

45 the detector used for imaging infrared thermography, temperature sensitivity is limited by the overall system noise.
Temperature sensitivity also depends on the materials used and on the emissive and reflective properties of the last
interface between the imaged object and air in front of the detector.

[0006] There are also numerous methods of optically monitoring biological interactions and/or chemical reactions
based on the measurement of refractive index. Besides standard refractive index-based methods, such methods in-

50 elude evanescent wave-based methods using for example, surface plasmon resonance or optical resonant structures
such as grating couplers and resonant mirrors. For example, United States Patent Number 5,738,825, the entire con-
tents of which are incorporated herein by reference, describes an optical biosensor including a detection cell including
a transparent base plate and a sample plate on the base plate. The sample plate has a matrix of wells to receive a
sample, and the base plate includes a diffraction grating and a waveguiding film to incouple incident light into the

55 waveguiding film adjacent the bottom of the well structure. The incoupled light field generates a diffracted light field to
enable detection of a change in the effective refractive index of the waveguiding film.

[0007] In the optical detection methods described above, the biological interactions monitored are performed in a
liquid medium, typically an aqueous medium, in contact with the sensing area. One limitation of these optical detection

EP 1 262 764 A1

methods is that the refractive index of the sensing structure may vary with temperature.

[0008] Another limitation is that there may be refractive index variations among various locations of the sensing
structure. For example, when the sensing structure is a microplate containing a plurality of wells (e.g., 6, 24, 96 or 384
wells), there can be temperature variations in different wells of the same microplate. This temperature variation in

5 different wells may be 3 °C or higher. In addition, for systems that monitor temperature in a liquid medium, the variation
of refractive index related to the biological event is further masked by the refractive index variations of the liquid medium
itself. Different volumes of liquids in different wells affect the path length of the optical signal, leading to further signal
variations in optical detection systems. This difference in volume can be due to the fact that a different volume of liquid
has been dispensed in an individual well, or due to evaporation of the liquid from the wells. These well-to-well volume

10 differences either generate signal variations or must be compensated by some other means. One commercially avail-
able detector available from Molecular Devices, Sunnyvale, California corrects for well volume differences. However,
it would be advantageous and less complicated if a system did not require corrections in the volume of liquid in each
well to simply the analysis of samples in microplates. Furthermore, there is no known method or apparatus that corrects
for refractive index changes due to temperature changes in the local sensing area.

15 [0009] Biological interactions monitored by the methods referenced above typically translate in refractive index var-
iations ranging from 10 -2 down to 10 5 and lower. Therefore, the measurements must be performed in an environment
in which temperature is rigorously controlled, preferably with temperature variations lower than 0.1 °C to preserve
accuracy and sensitivity of the measurement.

[001 0] There is a need to provide devices, methods and systems capable of performing fast and reliable high through-
20 put screening of cells and biomolecules. It would be desirable to perform the high throughput screening using standard
instrumentation and a relatively simple method, which would facilitate screening drug candidates for their interaction
with another compound. In addition, for devices, methods and systems that utilize changes in refractive index to monitor
metabolic changes in cells and interactions between and among biomolecules, it would be useful to provide the capa-
bility to monitor for and compensate for temperature-induced changes in refractive index.

SUMMARY OF INVENTION

[0011] Accordingly, the present invention generally provides methods, devices and systems for assaying samples,
particularly samples including biomolecules or cells. One aspect of the invention involves a substrate, for example, a

30 microplate for assaying samples including a frame forming sidewalls of at least one well and a bottom portion that
forms a bottom of at least one well. According to this aspect, the bottom portion includes a fluorescent material in
thermal communication with the at least one well. In a preferred aspect, the fluorescence of the fluorescent material
changes as the temperature of the at least one well changes. Desirably, the fluorescent material is operative to produce
a change in fluorescence to detect a chemical reaction, a biomolecular reaction, or a metabolic response of a cell.

35 [0012] A chemical reaction could simply involve detecting whether two different reactants produce an endothermic
or exothermic reaction, such as the mixture of potassium hydroxide and water. An example of a biomolecular reaction
is the binding of a biomolecule to another compound. Such binding typically results in a metabolic change in a biomol-
ecule, which produces either a positive or negative heat of reaction. A metabolic response of a cell may be produced,
for example, when a cell or cell fragment is contacted with a serum, which can be detected by monitoring the temper-

40 ature of the cell to determine if there is a change in temperature. By monitoring the fluorescence of a fluorescent
material having a temperature dependent fluorescence, the presence or absence of a reaction or metabolic change
can be detected on a sample substrate such as a microarray of biomolecules or cells or a microplate well that incor-
porates such a fluorescent material.

[0013] According to one aspect of the invention, the fluorescent material is in the form of a film forming a layer
45 adjacent the bottom portion of the substrate. Preferably, according to this aspect, the film has a thickness less than
about 50 microns. In another aspect, in which the substrate is a microplate, the bottom portion of the microplate has
a bottom surface, and the layer is adjacent the bottom surface, preferably the surface contacting a fluid contained in
the well. In still another aspect of the invention, the fluorescent material is embedded in the bottom portion of the
substrate. According to another aspect of the invention, the fluorescent material includes a rare-earth chelate. A par-
50 ticularly preferred rare earth chelate is EuTTA. Other preferred fluorescent materials include Rhodamine B, Erythrosin
B or terthiophene. Other potential fluorescent materials that may be used according to the present invention, include,
but are not limited to, EuFOD, EuTFC, TbFOD, EuBA, EuTHD, EuHFC, EuDBM, EuTA, EuTFA, EuDCM, TTED, TbTTA,
TbTFA, TbBA, TbTHD, TbAA, and combinations thereof.

[001 4] Another aspect of the invention relates to a substrate including a biomolecule, a cell or a cell fragment bound
55 to the surface of the substrate and a fluorescent material in thermal communication with a surface of the substrate.
The fluorescent material has a temperature dependent fluorescence. Preferably, the substrate includes a microarray
of biomolecules, cells, or cell fragments on a surface thereof.

[0015] Another aspect of the invention involves a method of detecting a chemical reaction, a biomolecular reaction

EP 1 262 764 A1

or a metabolic change in a cell. The method includes providing a reaction substrate such as a microplate or a microarray
chip including a fluorescent material. According to this aspect, the fluorescence of the fluorescent material changes
as the temperature of the fluorescent material changes. The method further includes placing a chemical, a biomolecule
or a cell in reactive contact in or on the reaction substrate and monitoring the fluorescence of the fluorescent material.

5 [0016] As noted above, when two chemicals react, or when a biomolecule and a second compound react, such as
when a target molecule and a receptor bind, or when there is a metabolic change in a cell, a positive or negative thermal
energy is created. This thermal energy change can be detected by monitoring fluorescence of a material having a
fluorescence that changes with changing temperature. Thus, according to another aspect of the invention, the method
may further include correlating a fluorescence reading with chemical or biomolecular reaction or a metabolic change

io in a cell by, for example, determining the change in temperature based on the change in fluorescence.

[0017] The present invention further provides a method of screening biomolecular or cellular assays. According to
this aspect of the invention, the method involves providing biomolecules or cells in an array of locations, such as in a
microplate or a microarray chip used for high throughput screening of biomolecules or cells. This aspect further involves
placing a compound in reactive contact with the biomolecules or cells in at least one of the locations and detecting the

15 temperature change in the at least of one of the locations by detecting the change in fluorescence of at least one of
the locations. Preferably, at least one of the array of locations contains a fluorescent material, the fluorescence of which
changes with temperature.

[0018] Another aspect of the invention relates to a system for high throughput screening of biomolecules or cells.
The system includes a sample holder including an array of locations, the sample holder including a fluorescent material,

20 the fluorescence of which changes with temperature. The system further involves providing a structure, method or
device for contacting the biomolecules or cells with a compound in at least one of the array of locations. The system
also includes a measurement device, for example, a fluorescence microthermal imaging device, for detecting the
change in fluorescence of the fluorescent material as the temperature in at least one of the locations changes.
[0019] Another aspect of the invention relates to an optical sensing system including a substrate in contact with a

25 fluid containing a biomolecule or a cell, a waveguide associated with the substrate, a light source, a light detector and
a fluorescent material, the fluorescence of which changes with changing temperature. According to this aspect of the
invention, the waveguide may include one or more planar waveguides, optical fibers, grating structures, or combinations
thereof. The optical sensing system may further include a processor for determining temperature changes in accord-
ance with the changing in fluorescence of the fluorescent material. In a preferred aspect, the processor is operative to

30 receive an optical signal and correlate changes in temperature with changes in the refractive index of the substrate
and/or the fluid. Preferably, the processor is operative to adjust the optical signal in accordance with the correlated
change in refractive index of the fluid and/or the substrate to provide a compensated optical signal for the temperature
dependent refractive index change of the substrate or the fluid.

[0020] Still another aspect of the invention relates to a method for analyzing substances proximate a sensing area
35 of a surface. The method includes the steps of detecting a light signal generated proximate the sensing area, measuring
the temperature proximate the sensing area, measuring the refractive index of the sensing area, and determining the
change in refractive index of the sensing area due to the change in temperature and adjusting the light signal in ac-
cordance with the change in refractive index. According to this aspect, the sensing area may include a substrate and
a fluid containing a cell or a biomolecule in contact with the substrate. A suitable substrate may include a microplate,
40 a microarray chip or a microfluidic chip. Preferably, a fluorescent material having a temperature-dependent fluorescence
is proximate the sensing area.

[0021 ] The invention provides a relatively simple and flexible method using standard instrumentation to detect chem-
ical reactions, biomolecular reactions and metabolic changes in a cell, which facilitates high throughput screening of
biomolecules. Additional advantages of the invention will be set forth in the following detailed description. It is to be
45 understood that both the foregoing general description and the following detailed description are exemplary and are
intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

50 [0022]

FIGS. 1A-1C show microplate structures having a fluorescent material associated with the microplates according
to the invention;

FIGS. 2A-2B show schematic representations of systems for monitoring the change in temperature in a sample
55 holder having an array of locations containing biomolecules;

FIG. 2C shows a schematic representation of a system for monitoring the change in temperature in a sample
holder having an array of locations containing biomolecules and correlating the changes in temperature with re-
fractive index change;

EP 1 262 764 A1

FIG. 3 is graph showing the absorption and emission spectra of a EuTTA/PMMA film on a microplate;
FIG. 4 is a graph showing the temperature dependence of the emission at 614 nm of a EuTTA/PMMA film;
FIG. 5 is a graph showing the variation of fluorescence signal with varying temperature over time;
FIG. 6 is a graph showing the variation of refractive index with changing temperature for water;
5 FIG. 7 is a graph showing temperature dependence of the emission at 440 nm of a terthiophene/PMMA film;

FIG. 8 is a graph showing the change in fluorescence of EuTTA/PMMA film in coated microplate well containing
KOH upon dilution with water versus time; and

FIG. 9 is a graph showing the change in fluorescence of EuTTA/PMMA film in coated microplate well containing
Kl upon dilution with water versus time

DETAILED DESCRIPTION

[0023] The invention relates to assay methods, devices and systems for monitoring biological or chemical interac-
tions, by providing means to monitor the temperature in at least one location in an array of locations. Additionally, the

15 invention is compatible with optical detection principles known in the art such as fluorescence intensity, fluorescence
anisotropy, fluorescence energy transfer, time resolved fluorescence, luminescence and combinations thereof, which
may be used in the determination of interaction characteristics that do not have an effect on temperature of a location.
According to the present invention, the interactions between and among chemicals, cells and biomolecules, can be
detected by monitoring the temperature of at least one, but preferably more than one location in real time. Such tem-

20 perature monitoring provides the ability to detect and measure those interactions between biomolecules and a second
compound associated with heat generation or consumption.

[0024] A preferred aspect of the invention utilizes a substrate having a fluorescent material associated with the sub-
strate. As used herein, the term "substrate" refers to a sample holder or container suitable for use in the measurement
of interaction between biomolecules in an array of locations. Such as substrate, can include, for example, a microf luidics
25 chip, a microplate or a microarray chip suitable for use in high throughput screening of biomolecules. The fluorescent
material has a fluorescence that changes with changing temperature.

[0025] The fluorescent material may be associated with a substrate in a variety of ways. According to one aspect of
the invention, the fluorescent material may be mixed with a solvent and applied to the substrate by spraying, dipping,
coating, brushing and other methods that can form a uniform and reproducible coating on a substrate, which can be
30 made from a variety of materials. The fluorescent material may be part of a composite material for optimum heat
capacity and thermal conductivity. Structured composites can also be used to induce anisotropy in thermal conductivity
to improve the heat transfer between the sample and the temperature sensitive material.

[0026] Alternatively, the fluorescent material can be manufactured in the form of a sheet or a film, which can be
placed in contact with the substrate. Another way of associating the fluorescent material may be by incorporating the

35 material into the structure of the substrate. For example, the fluorescent material could be impregnated into the material
used to manufacture the substrate. Such impregnation methods are known in the art of manufacturing substrates such
as microplates made from polymeric materials. As one example, a dye compound including the fluorescent material
could be dispersed in a matrix of the material used to make the substrate. The matrix could be an organic matrix such
as a polymeric material or an inorganic matrix made of solgel materials. The fluorescent material would not necessarily

40 have to be incorporated into the entire structure of the substrate, and preferably, only a portion of the substrate would
have the fluorescent material incorporated therein. For example, if the substrate is a microplate, it may be desirable
for only the bottom portion of the microplate to have the fluorescent material incorporated therein. Alternatively, a
temperature sensitive film can be made by forming a thin film of a matrix material and a fluorescent die using techniques
such as casting, rolling, extruding and the like. The particular means of associating the fluorescent material with the

45 substrate will depend at least upon the type of substrate desired and the type of material used to manufacture the
substrate. Such substrate materials include, but are not limited to glass, quartz, silica, ceramics, metals, polymeric
materials, and combinations thereof.

[0027] According to the present invention, the fluorescent properties of the fluorescent material associated with the
substrate depend on temperature. Temperature dependent properties of the fluorescent coating or film can include the
so emission intensity at a given wavelength or over a range of wavelengths or the spectral characteristics of the emitted
light.

[0028] Fluorescent dye compounds exhibiting high temperature dependence of their fluorescent properties are
known in the art. For example, Europium (III) Thenoyltrifluoroacetonate trihydrate (EuTTA) shows a decrease of fluo-
rescence intensity with increasing temperature. Alternatively, some compounds exhibit the reverse effect with increas-
55 ing fluorescence intensity when temperature increases. The fluorescent layer or film can be made essentially trans-
parent except for the portion of the spectrum where the fluorescent material is light absorbing. It will be understood,
that a variety of fluorescent materials may be used in accordance with the present invention. EuTTA is a particularly
preferred material. Other preferred materials include Rhodamine B, erythrosine B or terthiophene. Examples of other

EP 1 262 764 A1

candidate materials that may be used in accordance with the invention include, but are not limited to, EuFOD, EuTFC,
TbFOD, EuBA, EuTHD, EuHFC, EuDBM, EuTA, EuTFA, EuDCM, TTED, TbTTA, TbTFA, TbBA, TbTHD, TbAA, and
combinations thereof.

[0029] The thickness and the concentration of luminescent dye of the temperature sensitive coating or film have an
5 influence on the temperature sensitivity of the coating or film. In order to optimally detect the heat generation or con-
sumption produced by the reaction in a sample, the heat capacity and thermal conductivity of the materials (coating
and substrate or film) should be optimized. Therefore, the overall thickness of the temperature sensitive part of the
substrate, such as a microplate or microarray chip, in thermal communication with the sample (e.g., bottom of the well
+ coating or film) are preferably thin (on the order of a fifty micrometers or less). For a given coating or film thickness,
10 the concentration of the dye is preferably adjusted at the upper limit where absorption versus concentration relationship
starts to depart from linearity.

[0030] Preferably, according to the present invention, microplates having thin portions at the bottom of each well are
used. The thin bottom portion of a substrate can include, but is not limited to transparent polymers, polystyrene, poly-
propylene, UV transparent film, glass, metal and combinations thereof. It may be desirable to include a metal film in

15 the bottom portion of the substrate such as aluminum foil because metal films have high thermal conductivity.

[0031] It will be appreciated that the fluorescent material can be positioned in a variety of locations with respect to
a substrate. For example, as shown in FIG. 1 A, a microplate 10 is shown having a bottom portion 12 having a bottom
surface 13. A fluorescent film 14 is positioned on the bottom surface of the bottom portion of the microplate. In an
alternative embodiment shown in FIG. 1 A, the microplate 10 having a bottom portion 12 includes a fluorescent film 14

20 located between microplate wells 16 and the bottom portion 12. In still another embodiment microplate 10 can include
a bottom portion 12 incorporating the fluorescent material. As discussed above, such incorporation can involve im-
pregnating the material used to make the bottom portion of the microplate, or alternatively, the bottom portion could
be doped or coated with the fluorescent material.

[0032] In embodiments in which the substrate is a microplate, the fluorescent material can form continuous layer
25 over the entire bottom portion of the substrate used to close wells or the fluorescent material may be associated with
individual wells by coating or doping the individual wells with the fluorescent material.

[0033] A wide variety of fluorometers and luminometers are used in biological assays or high-throughput screening
of drug compounds by end-point or real-time reading of microtiter plates. FIGS. 2A and 2B show schematics of an
exemplary temperature monitoring system according to the invention. FIG. 2A illustrates a setup including a microplate

30 20 having a fluorescent coating 21 utilizing a light source 22 that produces a light beam 23 directed at the fluorescent
material 21 associated with the microplate 20. A detector 24 detects the light emitted by the fluorescent material as-
sociated with the microplate 20. A processor associated with a central processing unit (not shown) correlates the
fluorescence with a temperature reading and provides a temperature indication in the wells of the microplate. FIG. 2A
shows a setup in which the light source and the detector are positioned above the microplate wells. FIG. 2B shows a

35 setup in which the light source 22 and the detector 24 are positioned below the microplate wells. It will be understood
that the invention is not limited to a particular detector and light source configuration, and other configurations are
within the scope of the invention. Additionally, while FIGS. 2A and 2B show a microplate, it will be understood that any
suitable substrate for chemical or biological analysis can be utilized and incorporate a fluorescent material according
to the present invention. Accordingly, the substrate could be a cuvette, a microarray, a microf luidics device or any other

to suitable substrate for processing chemical and biological materials.

[0034] According to the present invention, a substrate having a fluorescent material is used in such instruments by
interposing a portion of the substrate incorporating the fluorescent material with an excitation beam and by detecting
the emitted light at a particular wavelength or over a range of wavelengths. The emitted light property, for example,
intensity at peak emission wavelength, provides a direct measure of the temperature of the coating or film at the bottom

45 location being measured. This temperature can then be correlated to determine whether a biomolecule has reacted
with a compound to create a positive or negative heat of reaction.

[0035] The invention is particularly useful for performing cell-based assays for detecting and monitoring metabolic
changes induced by chemical or biochemical stimuli in cells. For example, if a microplate having a bottom portion
including a fluorescent layer is utilized in accordance with the present invention, cells may be dispensed in each well

50 with a volume of culture or nutritive medium. After the measurement instrument is thermally equilibrated, the temper-
ature in at least one microplate well is real-time monitored by measuring the optical response of the temperature
sensitive film or coating. By dispensing and mixing different compounds in each well and monitoring the temperature,
it is possible to identify compounds that have an effect on thermogenic processes of the cells by impacting their me-
tabolism. This protocol can be used, for example, to identify agonists and antagonists of therapeutically important

55 membrane receptors such as G-protein coupled receptors, tyrosine kinase receptors, or nuclear receptors, inhibitors
of enzymatic reactions and the like.

[0036] It is also within the scope of the invention to utilize a high density microarray chips for high throughput screening
of biomolecules using an imaging system. As used herein, the term biomolecule includes a variety of biological mate-

EP 1 262 764 A1

rials, including, but not limited to amino acids such as DNA and RNA, peptides, proteins, oligonucleotides, lipids, or
portions of cells. As noted above, because a typical target for drug action is with and within the cells of the body, cells
themselves can provide a useful screening tool in drug discovery when combined with sensitive detection reagents. It
thus would be useful to have a high throughput, high content screening device to provide high content spatial information

5 at the cellular and subcellular level as well as temporal information about changes in physiological, biochemical and
molecular activities. For example, if the chip substrate having a portion including a fluorescent layer or film is utilized
in accordance with the present invention, cells may be dispersed and attached to the substrate in a high spatial density
array. The optical response of the temperature sensitive coating or film can be measured using an imaging fluorometer
including a charge coupled device, allowing multiplexed detection of the thermogenic effects of multiple compounds

10 on identical cells or biomolecules. Alternatively, multiplexed detection of the thermogenic effects of the same com-
pounds on different cells or biomolecules can be performed.

[0037] Further modifications of the invention could include combining temperature monitoring with another meas-
urement such as time resolved fluorescence to provide additional characterization of the biological system preferably
in the same instrument used to monitor the temperature.

15 [0038] Another aspect of the invention relates to compensating for temperature variations by using a fluorescent
material having temperature dependent optical properties. Preferably, the fluorescent coating having temperature de-
pendent optical properties is positioned proximate to the sensing area and is used to monitor refractive index changes
associated with the biological or chemical samples. The temperature compensation system and method involves the
measurement of the luminescent property of the coating that in turn gives a measure of the local temperature of the

20 sensing area where the refractive index is being measured. Refractive index variations of the sensing area, which may
include the substrate and/or the fluid containing the biological or chemical sample, can be calculated and compensated
for in real time by adjusting for the local temperature contribution to the refractive index variations of the sensing area.
In other words, if the refractive index variation over a temperature range of the substrate and the fluid are known, the
variation in refractive index can be compensated for by adjusting the optical signal obtained for these variations.

25 [0039] For example, the temperature dependence of the refractive index of water, which is routinely used for chemical
and biological processing, is illustrated in FIG. 6. The variation of the refractive index of water with temperature is thus
described by the following:

30 An/AT = -(4X10" 5) -(2X10' 6 )T (1)

at about 20 °C, An/AT = - 8 X 10' 5 degree" 1 (2)

at about 40 °C, An/AT = - 1 .2 X 10" 4 degree" 1 (3)

where n is the refractive index and T is the temperature. The system and the method of the present invention may be
capable of compensating for variations of refractive index produced by temperature variations in an aqueous medium
40 as low as 2.4 X 10" 6 at 20 °C (equation 2) and 3.6 X 10 6 at 40 °C (equation 3).

[0040] The luminescent material which may be in the form of a layer on a surface of a substrate or embedded in the
material forming the substrate according to the invention can be used advantageously in microplates, microarray chips,
microfluidic devices and microbioanalytical devices where temperature of fluids is monitored during device operation
or for detection purposes. For example, the temperature of nucleic acid samples undergoing PCR reaction or hybrid-
ization could be monitored in real time.

[0041] An example of an embodiment in which temperature and refractive index are monitored in real time is shown
in FIG. 2C. As shown in FIG. 2C, a substrate 40, which in the embodiment shown is a microplate containing a number
of wells 52, is in contact with a fluid 50 containing biomolecules, chemical or cells. The substrate 40 includes temper-
ature dependent fluorescent material 41 in association with the substrate. As in the earlier described embodiments
shown in FIGS. 2A and 2B, the fluorescent material 41 may be in the form of a film or layer, or it may be embedded
into the material that forms the substrate 40. The substrate may include a sensing area 54, which in the embodiment
shown in FIG. 2C is in the wells. It will be appreciated that the sensing area 54 is the area proximate to where the
chemical reaction, biological reaction or metabolic change of a cell occurs. According to this aspect of the invention,
a waveguide 56, which may be in the form of an optical fiber, a planar waveguide, a waveguiding film, a grating, a
mirror, an interferometer, or other appropriate waveguiding structure is in association with the substrate. In the embod-
iment shown in FIG. 2C, the waveguide 56 is a grating. The waveguide may also include a combination of waveguiding
structures. For example, the waveguide may include a waveguiding film and a grating in combination as disclosed in

EP 1 262 764 A1

United States Patent Number 5,738,825, the entire specification of which is incorporated herein by reference. As shown
in United States Patent Number 5,738,825, a waveguiding film and a separate diffraction grating are associated with
each individual well in a microplate. Appropriate waveguiding films include, but are not limited to metal oxide materials
such as silica, titania, titania-silica, alumina, and other suitable waveguiding materials.

5 [0042] Still referring to FIG. 2C, a light source 42 directs a light beam 43 toward the sensing area 54. Waveguide 56
directs the light beam towards detector 44 generated proximate the sensing area 54. A processor 58 in communication
with the detector is associated with a central processing unit (not shown). The detector 44 will read the fluorescence
generated by the fluorescent material proximate the sensing area 54, and the processor 58 determines temperature
changes based on the fluorescence signal, which may, for example, be an increase or decrease in fluorescent intensity.

w [0043] According to another aspect of the invention, the detector 44, or a separate detector (not shown) , may measure
the refractive index of the sensing area, which may include the fluid and the substrate surface. The processor 58 (or
a separate processor) is operative to adjust the refractive index reading based on the change in temperature in the
sensing area. For example, if the refractive index of the substrate material and the fluid are known over a temperature
range, this information can be used to compensate the change in refractive index for the change in temperature and

is provide an compensated or corrected reading of the refractive index proximate the sensing area.

[0044] It will be understood, of course, that the temperature dependent fluorescent materials of the present invention
can be utilized to correct a variety of temperature dependent measurements. Accordingly, the invention is not intended
to be limited to the correction of measurements of refractive index. For example, the temperature dependent fluorescent
materials can be utilized to compensate temperature variations in Mach-Zehnder interferometers and other interference

20 structures such as photon sieves. Mach-Zehnder interferometers are well known. One type of photon sieve is described
in United States Patent Number 5,272,332, the entire contents of which are incorporated herein by reference. Briefly,
the photon sieve describe in United States Patent Number 5,272,232 can act a laser discrimination filter based on
temporal coherence. The structure comprises a multilayer device wherein the optical thickness of each layer is greater
than the coherence length of the ambient light, but still much smaller than the coherence length of the laser light of

25 interest. The spectral response of the photon sieve described in United States Patent Number 5,272,232 becomes
dependent on the degree of temporal coherence of the incident light. If white light strikes the filter, multi-beam inter-
ference will not occur because of its short coherence length. Thus, the photon sieve described in United States Patent
Number 5,272,232 acts like a stack of partially reflecting mirrors. If the laser light strikes the filter, multi-beam interfer-
ence will still take place because of the long coherence length of the laser light. This causes the device to have different

30 transmitting characteristics for laser light and white light. It will be understood, of course, that other photon sieve struc-
tures can be used in accordance with the present invention. For example, a structure including a number of submicron-
sized holes arranged in an optically active (e.g., a filtering or interfering) structure can be provided and temperature
compensation can be accomplished using the fluorescent materials according to the present invention.
[0045] In devices utilizing Mach-Zehnder devices or photon sieves in contact or filled with a fluid, the temperature

35 effect on refractive index of the fluid can be corrected by utilizing the fluorescence reading. Another important aspect
is the time dependence of the temperature drifts that are likely to be different from the kinetics of the events to be
measured. The fluorescent materials of the present invention can be used to provide a real time correction of the
temperature drifts in the fluid and compensate for the difference between the kinetics of the events being measured.
[0046] In another aspect of the invention, a capillary or film containing at least one capillary can include the temper-

40 ature dependent fluorescent materials of the present invention. For example, a thin coating (e.g., 20 microns or less)
of a porous structure including the temperature sensitive coating can be provided. Alternatively, a porous membrane
made from a suitable material, such as, for example, PMMA, can be filled with a temperature dependent fluorescent
material. Either one of these structures can be utilized as a membrane for cell cultures and detection of a metabolic
change in a cell such as a metastatic invasion, as the cell migrates through the capillary and the temperature of the

45 capillary adjacent during migration is monitored.

[0047] The process steps of the invention described above can be carried out using conventional equipment known
in the art, e.g., equipment commonly found in a bioanalytical laboratory. For example, the principle of the present
invention can be utilized with conventional analysis equipment to detect temperature changes associated with a change
in fluorescence. Without intending to limit the invention in any manner, the present invention will be more fully described

so by the following examples.

EXAMPLES

Example 1

EuTTA/PMMA Coating in Microplate Wells

[0048] EuTTA and PMMA (polymethylmethacrylate) were dissolved in a highly volatile solvent. For example, 2% w/

EP 1 262 764 A1

w EuTTA, 2% w/w PMMA, and 96% w/w Methyl ethyl ketone (MEK) were mixed in a container. A 96 well microplate
having a transparent polypropylene bottom (Corning, Inc. (catalog #9520), Corning, New York) was obtained, and an
appropriate volume of the solution was deposited with a micropipette at the center of each plate well. To coat the bottom
of a polypropylene plate with EuTTA/PMMA/MEK, approximately 15 uJ of solution was used. A film was formed by
5 solvent evaporation at room temperature and pressure. The coated plate was UV cured (365nm, 1 J/cm 2 ) to stabilize
the coating.

Example 2

10 Absorption Spectrum of EuTTA/PMMA Coating

[0049] The absorption spectrum of the coating deposited on the microplate wells in Example 1 was measured every
2 nm with a SpectraMax® Plus (Molecular Devices Corporation, Sunnyvale, California) UV/VIS microplate spectro-
photometer. The absorption spectrum extended from 200 to 400 nm and showed a maximum at 346 nm. The emission
15 spectrum ranged from 500 to 650 nm and showed a maximum at 614 nm. The absorption and emission spectra are
shown in FIG. 3.

Example 3

20 Temperature Dependence of Fluorescence Signal from EuTTA/PMMA Coating

[0050] The fluorescence signal emitted by the coating deposited in Example 1 was measured at different tempera-
tures. The microplate was heated and the fluorescence was measured at an emission wavelength of 614 nm using a
SpectraMax® Gemini (Molecular Devices Corporation) dual-scanning microplate spectrofluorometer at an excitation
25 wavelength of 355 nm over a temperature range from about 25 °C and 34 °C. The results in FIG. 4 show an approximate
3% decrease per °C for a 2% EuTTA/2% PMMA/96% MEK coating initial composition.

Example 4

30 Evaluation of the Limit of Detection

[0051] To evaluate the limit of detection, a noise measurement was done using a bottom read set-up as shown in
FIG. 2B using a Fluoroskan Ascent available from Labsystems. A microplate coated with EuTTA/PMMA was prepared
in accordance with Example 1 . One column (8 wells) of the microplate was filled with water and a kinetic measurement
35 was performed at temperatures ranging from 25 °C to 35 °C. For each temperature point, 50 fluorescence measure-
ments were performed at 20 seconds intervals. A graph of the results is shown in FIG. 5. Signal drift and signal noise
was calculated by assimilating signal drift to a straight line and calculating its slope. The slope was equal to -0.0073/°C.
The fluorescent signal measured was corrected according to the following formula (corresponding to the slope observed
on the curves):

Signal corrected =Signal raw + 0.0073 X Temperature

[0052] The standard deviation of 50 data points was calculated to be 0.285. Knowing that the fluorescent signal
45 varies 9.4241 units per degree centigrade, the noise and also the method sensitivity was calculated to be 0.285/9.4241
or equal to 30 X 10 3 °C.

Example 5

so Terthiophene/PMMA Coating in Microplate Wells

[0053] Terthiophene and PMMA (polymethylmethacrylate) were dissolved in a highly volatile solvent. For example,
2% w/w terthiophene, 2% w/w PMMA, and 96% w/w Methyl ethyl ketone (MEK) were mixed in a container. A 96 well
microplate having a transparent polypropylene bottom (Corning, Inc. (catalog #9520), Corning, New York) was ob-
55 tained, and an appropriate volume of the solution was deposited with a micropipette at the center of each plate well.
To coat the bottom of a polypropylene plate with EuTTA/PMMA/MEK, approximately 15 uJ of solution was used. A film
was formed by solvent evaporation at room temperature and pressure. The coated plate was UV cured (365nm, 1 J/
cm2) to stabilize the coating.

EP 1 262 764 A1

Example 6

Temperature Dependence of Fluorescence Signal from Terthiophene/PMMA Coating

5 [0054] The fluorescent signal emitted by the coating deposited in Example 5 was measured at an emission wave-
length of 440 nm with an excitation wavelength of 355 nm over a temperature range from 30 °C to 38 °C. The results
are shown in FIG. 7 and show an approximate 1 0% fluorescence signal increase per degree centigrade between about
34 °C and 37 °C.

[0055] Others dyes such as Rhodamine B and Erythrosin B were also used and showed that a change in fluorescence
10 associated with a temperature change in a reaction chamber could be monitored.

Example 7

Detection of Chemical Reactions

[0056] To demonstrate the principle that the change in fluorescence of a fluorescent material could be used to detect
a chemical reaction between two compounds, an experiment was performed with chemicals having known endothermic
or exothermic dissolution behavior in water.

[0057] First, eight wells of a 96 well microplate available from Corning, Inc., Corning, NY were coated with a solution
20 of a EuTTA/PMMA in accordance with Example 1 above. The coated wells of the microplate were filled with 200 ul of
water. KOH was added to seven of the eight coated wells, and one well was used as a reference well. The fluorescence
signal from the wells was measured at an excitation wavelength of 355 nm and an emission wavelength of 614 nm
over a 15 minute interval. FIG. 6 shows the temperature increase, represented by the change in fluorescence signal,
due to the exothermic dissolution of KOH in water versus the reference well. A kinetic measurement monitoring the
25 corresponding wells fluorescence signal with time was started immediately after the chemical addition at 25 °C. As
expected, in case of an exothermic phenomenon, the signal decreases immediately and then goes back to its initial
value, proving that the temperature increases due to the chemical addition and decreases progressively down to its
initial value after the dissolution is complete.

[0058] Similarly, with an endothermic phenomenon as shown in FIG. 9, the fluorescence signal was increasing due
30 to the chemical addition and went back progressively to its initial value. For example, a 1 5% increase of signal was
observed during the dissolution of 30 mg iodide potassium (Kl) in 200jil water. This example shows that it is possible
to monitor and detect a chemical reaction between two compounds.

Example 8

Detection of Metabolic Response in a Biomolecule

[0059] To make in vitro cell culture, animal serum is usually added to the basic medium to provide to the cells nutri-
ments and growth factor. A deprivation of this kind of serum leads to a slowing down of the cell metabolism. If serum
40 is added after a deprivation period, the cell metabolism is reactivated and heat is generated.

[0060] The present experiment detects the response, of CEM cells to serum addition in their culture medium after a
serum deprivation period.

[0061] The culture medium is prepared as follows (each component is provided by GibcoBRL Life Technologie):

45 RPMI: 89.8%

Veal Fetal Serum: 9%

Antimycotic Antibiotic: 0.9%

HEPES: 0.1%

Sodium Pyruvate: 0.1%
50 Glucose: 0.1%

[0062] CEM cells are cultivated in the medium described above at 37 °C in an incubator with 5% of C0 2 atmosphere.
Three to four hour before the beginning of the experiment, they are centrifuged and the medium is eliminated and
replaced by a fresh one that does not contain any Veal Fetal Serum. The sample is divided in two parts of same volume:
55 to the first one, veal fetal serum is added (these cells are not serum deprived) to reach 9% of serum in the total medium
composition while to the second one, the same volume of medium (without veal fetal serum) is added. The two samples
are put in the incubator again 3 to 4 hours. After this period of time, they are centrifuged, their media are eliminated
and replaced by fresh media (with serum in the first case and without serum in the second case). They are counted

EP 1 262 764 A1

and dispenses in the wells of a 384 well-microtiterplate (provided by Corning, Inc. reference 371 2). The total dispensed
volume per well is 50 jal and the cells number per well is 10 6 . Some additional wells are filled with the culture medium
only (some wells with the medium containing serum and some others with the medium without serum). The external
side of the microtiterplate bottom was previously treated with a EuTTA/PMMA coating using the same composition and

5 deposition method than in Example 1 .

[0063] The microtiterplate is installed in a bottom read fluorometer (Fluoroskan Ascent) temperature regulated at
37 C. The fluorescence signal from the wells is measured with the fluorometer for 190 seconds (one measurement
every 10 seconds). Next, 40 |il of veal fetal serum at 37 °C is dispensed in every well, and the fluorescence signal
from the wells is measured again (1 measurement every 10 seconds).

io [0064] The fluorescence of the wells containing the serum deprived cells is expected to change after the serum
injection while the fluorescence of the other wells (non deprived cells, medium with serum and medium without of
serum) is not supposed to change, these later wells being the controls.

[0065] As can be seen in Table 1, the kinetic analysis revealed that 10 minutes after the serum injection, a 1.1%
fluorescence change is observed from the wells containing the serum deprived cells, meaning that a heat is generated

15 in these wells and detected. On the other hand, at the same time, the fluorescence signal from the wells containing
the control solutions have a value similar to this before serum injection, meaning that the addition of serum does not
generate any heat in these wells. After this 1 0 minutes, fluorescence from the wells containing the deprived cells tends
to slowly increase becoming closer and closer to its initial value (before serum injection). This is in accordance with a
stabilization of the cells after they get enough serum to reactivate their metabolism.

20 [0066] This result validates the use of temperature-dependent fluorescent coating to monitor cell metabolism acti-
vation.

Table 1

Fluorescence signal before serum injection
(au.)

Fluorescence signal 10 minutes after the
serum injection (au.)

Medium with serum

1178±3

1178±2

Medium without serum

1178+3

1178*3

Non deprived cells

1178±2

1178±3

Serum deprived cells

1176+2

1163±3

[0067] It will be apparent to those skilled in the art that various modifications and variations can be made to the
present invention without departing from the spirit or scope of the invention. For example, a variety of fluorescent
materials and combinations of fluorescent materials that exhibit a changing fluorescence with changing temperature
may be used in accordance with the present invention. For example, in addition to the fluorescent materials discussed
in the specification, other fluorescent materials including, without limitation, Rhodamine B, Erythrosin B, and terthi-
ophene can be used in accordance with the present invention. Thus, it is intended that the present invention cover
modifications and variations of this invention provided they come within the scope of the appended claims and their
equivalents.

Claims

1 . A microplate for assaying samples comprising:

a frame forming sidewalls of at least one well; and a bottom portion that forms a bottom of at least one well,
the bottom portion including a fluorescent material in thermal communication with the at least one well.

2. The microplate of claim 1 , wherein the fluorescence of the fluorescent material changes as the temperature of the
at least one well changes.

3. The microplate of claim 2, wherein fluorescent material is operative to produce a change in fluorescence sufficient
to detect a chemical reaction, a biomolecular reaction, or a metabolic response of a cell.

4. The microplate of claim 2, wherein the fluorescent material is in the form of a film forming a layer adjacent the
bottom portion of the microplate.

EP 1 262 764 A1

5. The microplate of claim 4, wherein the bottom portion of the microplate has a bottom surface, and the layer is on
the bottom surface.

6. The microplate of claim 2, wherein fluorescent material is embedded in the bottom portion of the microplate.

7. The microplate of claim 2, wherein the fluorescent material includes a rare-earth chelate.

8. The microplate of claim 7, wherein the rare earth chelate is EuTTA.

io 9. The microplate of claim 6, wherein the fluorescent material is selected from the group consisting of EuTTA, Rhod-
amine B, Erythrosin B, terthiophene, and combinations thereof.

10. The microplate of claim 5, wherein the layer is less than about 50 microns.

is 11. A substrate including a biomolecule, a cell, or a cell fragment in contact with a surface of the substrate and a
fluorescent material in thermal communication with a surface of the substrate, wherein the fluorescence of the
fluorescent material changes with changing temperature.

12. The substrate of claim 11, wherein the fluorescent material is selected from the group consisting of EuTTA, Rhod-
es amine B, Erythrosin B, terthiopene and combinations thereof.

13. The substrate of claim 11 , wherein the substrate includes a microarray of biomolecules, cells or cell fragments on
a surface thereof.

25 14. The substrate of claim 11, wherein the substrate comprises a porous structure including at least one capillary
through which the biomolecule, cell or cell fragment migrates, and the temperature of the portion of the capillary
adjacent the biomolecule, cell, or cell fragment is monitored.

15. The substrate of claim 11, wherein the biomolecule is a cell, and the temperature of the portion of the capillary
30 adjacent the cell is monitored to detect metabolic changes in the cell.

16. A method of detecting a chemical reaction, a biomolecular reaction or a metabolic change in a cell comprising:

providing a substrate including a fluorescent material, the fluorescence of which changes as the temperature
35 of the fluorescent material changes;

placing a chemical, a biomolecule, or a cell in contact with a second compound in or on the substrate; and
monitoring the fluorescence of the fluorescent material.

1 7. The method of claim 1 6, further comprising the step of correlating the fluorescence reading with a reaction between
40 the biomolecule and the compound.

18. A method of claim 17, further comprising the step of correlating involves comparing the light intensity of the fluo-
rescent material with a change in temperature.

45 19. The method of claim 16, wherein the substrate is a microplate, microfluidics device or a microarray chip incorpo-
rating a fluorescent film.

20. A method of screening biochemical assays comprising:

so providing biochemical molecules in an array of locations;

placing a compound in reactive contact with the biochemical molecules in at least one of the locations;
detecting the temperature change in the at least of one of the locations by detecting the change in fluorescence
of the locations.

55 21. The method of claim 20, wherein at least one of the array of locations contains a fluorescent material, the fluores-
cence of which changes with temperature.

22. The method of claim 21, wherein the array of locations is provided on a microplate.

EP 1 262 764 A1

23. The method of claim 21 , wherein the array of locations includes a microarray chip.

24. A substrate including an array of locations, the substrate being suitable for use in a high-throughput screening of
biomolecules or cells, the substrate including a fluorescent material, the fluorescence of which changes with tem-

5 perature.

25. A system for high-throughput screening of biomolecules or cells comprising:

a sample holder including an array of locations, the sample holder including a fluorescent material, the fluo-
10 rescence of which changes with temperature;

means for contacting biomolecules or cells with a compound in at least one of the array of locations; and
means for measuring the change in fluorescence of the fluorescent material as the temperature in at least one
of the locations changes.

15 26. An optical sensing system comprising:

a substrate in contact with a fluid containing a biomolecule or a cell;
a waveguide in association with the substrate;
a light source;
20 a light detector; and

a fluorescent material in association with the substrate, the fluorescence of which changes with changing
temperature.

27. The optical sensing system of claim 26, wherein the waveguide includes a planar waveguide, a waveguide film,
25 an optical fiber or a grating structure.

28. The optical sensing system of claim 26, wherein the system includes a photon sieve or an interferometer.

29. The optical sensing system of claim 27 further comprising a processor for determining temperature changes cor-
30 responding to the changing in fluorescence.

30. The optical sensing system of claim 29, wherein the processor is operative to receive an optical signal and correlate
changes in temperature with changes in the refractive index of the substrate and/or the fluid.

35 31 . The optical sensing system of claim 29, wherein the processor is operative to adjust the optical signal in accordance
with the correlated change in refractive index of the fluid and/or the substrate.

32. A method for analyzing substances proximate a sensing area of surface comprising:

40 detecting a light signal generated proximate the sensing area;

measuring the temperature proximate the sensing area;
measuring the refractive index of the sensing area; and

determining the change in refractive index of the sensing area due to the change in temperature and adjusting
the light signal in accordance with the change in refractive index.

33. The method of claim 32, wherein the sensing area includes a substrate and a fluid in contact with the substrate.

34. The method of claim 33, wherein the substrate is selected from the group consisting of a microplate, a microarray
chip and a microfluidic chip.

35. The method of claim 33, wherein the fluid contains a cell or a biomolecule.

36. The method of claim 35, wherein a fluorescent material having a temperature-dependent fluorescence is proximate
the sensing area.

37. The method of claim 36, wherein the fluorescent material is selected from the group consisting of EuTTA, Rhod-
amine B, Erythrosin B, terthiophene and combinations thereof.

EP 1 262 764 A1

38. The method of claim 37, wherein the fluorescent material is embedded in the substrate.

39. The method of claim 37, wherein the fluorescent material is in the form of a layer on a surface of the substrate.

5 40. The method of claim 39, wherein the substrate is selected from the group consisting of a microplate, a microarray
chip and a microfluidics chip.

41. The method of claim 36, wherein the substrate includes at least one capillary, and the fluorescent material is
adjacent the capillary.

10
15
20
25
30
35
40
45
50

EP 1 262 764 A1

FIG. 2C

EP 1 262 764 A1

240 340 440 540 640

Wavelength (nm)

FIG. 3

EP 1 262 764 A1

16000

| 15000
5 14000
§, 13000

2 12000
c

| 11000
o

i? 10000

y =

-490.39X 4

27644

R 2 = 0.97

27 29 31

Temperature (°C)

FIG. 4

EP 1 262 764 A1

Time (sec)

FIG. 5

y=-1l

i-tEx 2 ■

4E-05

x + 1.3

333

R 2 =

: 0.999

10 20 30 40 50 60 70 80 90 100
Temperature

fig. 6

EP 1 262 764 A1

7 000

Temperature (°C)

FIG. 7

dissolution of KOH in water

15 000

^ 13 000

150 200 250
Time (sec)

350

FIG. 8

Dissolution of Kl in water

17000

- Reference

500

FIG. 9

EP 1 262 764 A1

European Patent
Office

EUROPEAN SEARCH REPORT

EP 01 40 1376

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Full text of "USPTO Patents Application 10561839"

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